Analysis Of The Chemical Components And Multidimentional Fingerprint Of Root Of Isatis Indigotical Fort.

Banlangen is the dry root of lsatis indigotica Fort. which belongs to the family of Cruciferae. Banlangen is a famous traditional chinese medicine for antipyretic and detoxifying purposes and is often used for virosis and bacterial infection diseases in clinical. In this thesis, the analysis of the components of Banlangen by LC/DAD/ESI/MS/MS technique, goitrin preparation and identification, content determination, fingerprint and multidimensional analysis of Banlangen and antivirus anti- endotoxin pharmacological experiments were investigated. The investigation is helpful for improving medicine quality and reasonably utilizing medicine resource.1. Analysis of the components of BanlangenWith the LC/DAD/ESI/MS/MS technique was used, the components of Banlangen were qualitative analyzed. Five compounds of Banlangcn were identified by the comparison of the mass spectra with corresponding authentic samples, the LC retention time and UV spectra and. There were cytidine, uridine, adenosine, guanosine and goitrin. Another 7 compounds, arginine, tyrosine, phenylalanine, isaindigodione, salicylic acid, indigoticalignanoside and hydroxyindirubin, were conjectured by their ESI/MS². The difference was compared between the Banlangen extracts of water and ethanol. The water extract was thought to be more comprehensive to reflect the components of Banlangen, and was chosen as the method to extract in this investigation.Baphicacanthus cusia (Nees) Brem. (ROBCB), another crucifer, was recorded as a breed by Chinese Pharmacopoeia in 1995. LC/MS technology was applied to analyze ROBCB and both the UV chromatograms and total ion chromatograms (TIC) revealed the difference between Banlangen and the root of ROBCB.2. Preparation and identification of goitrin from Banlangen The water extract of Banlangen was pre-isolated by the macroporoud adsorption resin (D-101) and was extracted by ethyl acetate. Semi-preparative RP-HPLC was used for final isolating and obtaining compound. Structure identification was performed by the analysis of EI-MS,¹H-NMR and ¹³C-NMR data and it was confirmed as goitrin. The developed method was simple, fast, high-yield, pure and low-cost. Goitrin should be regarded as one of the characteristic components for identification of Banlangen.3. Determination of nucleotide components and goitrin in BanlanganThe RP-HPLC method synchronously determined of uridine, adenosine and guanosine with equal elution was developed. The mobile phase was consisted of methanol and NaH₂PO₄-H₃PO₄ solution. The relative coefficient of uridine was 0.9996 in range of 0.0107～0.107 mg·mL^-1, and the average recovery was 101.0% with RSD 1.3%. The relative coefficient of adenosine was 0.9996 in range of 0.0101～0.101 mg·mL^-1, and the average recovery was 98.6% with RSD 1.1%. The relative coefficient of guanosine was 0.9995 in range of 0.0106～0.106 mg·mL^-1, and the average recovery was 98.8% with RSD 1.2%. A new method for improving qualitative criterion of Banlangen was provided.By this method, 19 samples were assayed and the contents of nucleotide components were much different from sample to sample. It was concluded that the germ plasm of this medicine, the terrain and the environment where they grew conduced the difference.Goitrin in Banlangen was determined by RP-HPLC with methanol-water (15: 85) as mobile phase. In range of 0.0107～0.1284 mg·mL^-1, the relative coefficient was 0.9999 and the average recovery was 102.2% with RSD 0.5%. Nineteen samples were determined. The method was simple, fast and veracious. There was no relativity in content between nucleotide components and goitrin.4. Manufacture technics optimization of Banlangen injectionUniform design method and single factor optimization were applied to optimize the manufacture technics of Banlangen injection. The size of medicine and steeping time were also studied. Finally, the new technics was established with practical manufacture considered.5. Fingerprint and multidimensional fingerprint analysis of BanlangenBoth gradient elution and equal elution were studied, and the fingerprints were developed respectively. In the LC-UV fingerprint analysis, 19 samples were classified by Hierarchical Cluster analysis, and the clustering results of gradient elution and equal elution had consistency. Two Common Modes were set up by computer aided similarity system and sample mixed, respectively. The Similarities compared with the two Common Modes were of consistency. In order to comprehensively reflect the components of Banlangen and estimate exactly, the fingerprint chromatogram by gradient elution was commended. With computer aided similarity system calculating Similarity, content determination should be considered to insure the medicine has complete components and regular content. The analysis results showed that sample with Similarity more than 0.9 was defined as the valid sample. The similarities of LC-UV chromatograms of medicine, intermediate and injection were compared, and the effect on fingerprints chromatograms used ODS columns with different sealed technique was studied.Six samples were involved to set up the LC-MS fingerprint of Banlangen, and the extracted ion chromatogram of adenosine was chosen as the reference peak. The similarities of LC-UV and LC-MS fingerprints of Banlangen were compared, and the evaluation results of the two methods were identical for most of the samples.6. Study on antivirus and anti-endotoxin activity of Banlangen1. Antivirus experiment Antivirus pharmacological experiment about the nucleotides was carried in vitro, and the nucleotides were proved to have activity on anti- influenza virus of Al and anti-adenovirusâ…¢. This study was the preparatory research for finding water-solubility antivirus components in Banlangen.2. anti-endotoxin experiment Banlangen was proved to possess obvious anti-endotoxin activity by 4 pharmacological experiments and can prevent and protect the fever heat and acute liver trauma induced by endotoxin.(1) Fever heat experiment of rabbit After Banlangen extract and crude goitrin extract were injected respectively, the fever heat induced by endotoxin could be prevented and protected. The temperatures of experimental rabbits were decreased and the economy was protected. The temperature decrease effect of Banlangen extract was better than crude goitrin extract.(2) Acute liver trauma experiment of mouse Mice shock and death induced by endotoxin were protected in different degree after Banlangen and goitrin extract were injected, and the protective effects were close to the effect of masculine group even better than it. The experimental result of mice acute liver trauma induced by endotoxin matched with the result of death rate experiment, which explained more the protective effect of Banlangen to liver.(3) Pharmacokinetic study of Banlangen extract and crude goitrin extract in rabbit was carried evaluated with goitrin. Plasma sample was extracted with dichloromethaneiso-prophl alcohol (4 : 1, v/v) and the residua after blown was dissolved by mobile phase. The mobile phase was consisted of acetonitrile - water (80 : 20, v/v) with paracetamol as internal standard at flow-rate 1.0 ml·min^-1. Diamonsil^TM C₁₈(4.6×250 mm, I.D.5μm) column was used. The linearity range was 0.123～4.90μg·mL^-1 and the lower limit of quantification was 0.123μg·mL^-1. The method was applicated to study the pharmacokinetics of 14 healthy rabbits after ventro-inject. For Banlangen extract, mean peak plasma levels (C_max) of 1.61±0.44μg·mL^-1 and T_max of 0.4±0.2 h were observed. The mean t_1/2 value of 1.67±0.52 h was obtained, AUC_0-t was calculated to be 4.24±1.09μg·mL^-1. For crude goitrin extract, C_max of 2.66±0.44μg·mL^-1 and T_max of 0.4±0.2 h were observed. The mean t_1/2 value of 1.88±0.48 h was obtained, AUC_0-t was calculated to be 7.51±1.40μg·mL^-1. Except the C_max different caused by different administration, there were no notable difference between T_max, t_1/2 and mean reservation time (MRT) analyzed by t test. By pharmacokinetics study, foundation was supplied for more research about ROCIIT.